The development of technologies allowing proteins to be synthesized at will are expected to contribute greatly, not only to the fields of the life sciences and biotechnology, but also to the design of nano-machines and the development of molecular devices in such engineering fields as neural computing. Currently, genetic engineering techniques for introducing cloned DNA into living cells are widely used for protein synthesis, but exogenous proteins that can be produced by these methods are limited to molecules that are able to survive the life support mechanisms of their host. Meanwhile, advances in organic synthesis technology have made automatic synthesizers common, but while peptides comprising a few dozen amino acids are routinely synthesized, chemical synthesis of higher molecular weight proteins is currently extremely difficult, due to limitations in terms of the yield, side reactions, and the like. Furthermore, there has been strong ethical criticism in Europe and the United States of conventional using living organisms to produce proteins, or to search for novel molecules, and there is a concern that international regulations will become even stricter.
Cell-free protein synthesis is a novel protein synthesis method capable of overcoming these problems, which attempts to make maximal use of the outstanding characteristics of living organisms by applying chemical procedures to the same. These methods provide biological systems for the translation of genetic information within artificial containers and, using nucleic acids which have been designed and synthesized as templates, reconstruct systems capable of incorporating the desired amino acids, including those which do not exist in nature. As these systems are not subject to the limitations of living organisms, it can be expected that an almost limitless range of protein molecules can be synthesized.
With regard to cell-free protein synthesis systems, it was reported 40 years ago that pulped cell sap retained the ability to synthesize protein, and various methods of doing this have been developed in the past. Currently, cell extracts derived from E. coli, wheat embryo, and rabbit reticulocytes are widely employed in protein synthesis and the like. Cell-free systems allow for rapid translation speeds of 10 peptide bonds per second, which is roughly equal to in vivo translation speeds, and excellent reaction characteristics in terms of translation accuracy, but in all cell-free systems, the period of time for which synthesis can be continued is short and the yield is extremely low, at a few micrograms to a few dozen micrograms per milliliter of reaction volume, which is approximately 1/100 to 1/1000 of the yield of living cells, making this an impractical protein synthesis method.
A major disadvantage of conventional cell-free protein synthesis systems was that synthesis efficiency was extremely low, but no direct studies have been made of the cause. This is because it was common knowledge in the field of biochemistry that activity in cell extracts prepared with artificial buffer from physical ground cells was somewhat lower.
The inventors have already shown, based on the findings of past research into ribosome inactivating toxins, that the extreme drop in protein synthesis activity seen in cell-free protein synthesis systems using wheat embryo extract were the result of a switch for an auto ribosome inactivation mechanism (cell suicide mechanism), which is programmed into the original cell as a defense mechanism against pathogenic microorganisms, and which is triggered by grinding the embryo (Madin, K. et al., Proc. Natl. Acad. Sci. USA, 97, 559-564 [2000]). Then, it was demonstrated that protein synthesis reactions using wheat embryo extract that was prepared by a novel method, wherein tritin activity and the like were eliminated from embryo tissues, exhibit good protein synthesis characteristics over a long period of time (Madin, K. et al., Proc. Natl. Acad.Sci. USA, 97, 559-564 (2000), JP-2000-236896-A).
However, there are problems with wheat embryo extracts prepared according to such methods in that, depending on the target protein (for example, DNA binding proteins such as transcription factors) by the influence of other inhibition factors meant that it was not always possible to achieve a sufficient yield.
Furthermore, conventional cell extracts for cell-free protein synthesis presented problems in terms of storage stability when solutions that contained the amino acids, energy sources, ions and the like, which are necessary for protein synthesis were added. For this reason, it was necessary to supply the cell extract and the solution containing the energy source and the like separately, requiring that the researcher mix these together with the translation template each time that an experiment was performed. Because of such problems as the necessity of performing these operations at low temperatures, the overall experimental work became difficult, which often caused protein synthesis to fail. Furthermore, such methods for supplying reagents for cell-free protein synthesis reactions are not suited for comprehensive synthesis of proteins from a multiplicity of genes and a major problem facing future robotization is the solution of these problems of complicatedness.